Compositions and Methods to Detect Non-Coeliac Gluten Sensitivity

ABSTRACT

Disclosed are compositions and methods to detect proteins associated with non-coeliac gluten sensitivity (NCGS). Such markers may be useful to allow individuals susceptible to NCGS to manage their food intake to avoid symptoms and further progression of disease.

RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.15/977,173, filed May 11, 2018, which claims priority to U.S.Provisional Patent Application No. 62/505,378, filed May 12, 2017. Thecontents of each of the above is incorporated by reference in itsentirety herein.

FIELD OF DISCLOSURE

The disclosure relates to methods and compositions for diagnosingnon-coeliac gluten sensitivity.

BACKGROUND

Coeliac disease is an autoimmune disorder with genetic, environmentaland immunological components. Symptoms of coeliac disease may betriggered by ingestion of wheat gluten and certain related proteins ofrye and barley. Such symptoms may include inflammation, villous atrophyand crypt hyperplasia in the small intestine. There are, however,certain individuals who experience a range of symptoms in response towheat ingestion without the characteristic serological or histologicalevidence of coeliac disease. The term non-coeliac gluten sensitivity(NCGS) and non-coeliac wheat sensitivity (NCWS) are generally used torefer to this condition.

The biological mechanisms behind NCGS are unknown and there are few, ifany, biomarkers that provide a reliable indication of this condition.Still, it would be helpful for individuals having susceptibility to NCGSto adjust their diet so as to avoid triggering an onset of symptomsand/or promoting further progression of the disease. Thus, there is aneed to develop and evaluate biomarkers for NCGS.

SUMMARY

The present disclosure may be embodied in a variety of ways.

In one embodiment, disclosed is a method to detect biomarkers associatedwith non-coeliac gluten sensitivity (NCGS) in an individual comprisingthe steps of: obtaining a sample from the individual; and measuring theamount of at least one of IL-8, IL-10, TNF-Alpha or total IgE protein inthe sample.

Other features, objects, and advantages of the disclosure herein areapparent in the detailed description, drawings and claims that follow.It should be understood, however, that the detailed description, thedrawings, and the claims, while indicating embodiments of the disclosedmethods, compositions and systems, are given by way of illustrationonly, not limitation. Various changes and modifications within the scopeof the invention will become apparent to those skilled in the art.

FIGURES

The disclosure may be better understood in view of the followingnon-limiting FIGURES.

FIG. 1 shows an example of a multi-node interaction network identifyingmarkers associated with NCGS.

DETAILED DESCRIPTION Terms and Definitions

In order for the disclosure to be more readily understood, certain termsare first defined. Additional definitions for the following terms andother terms are set forth throughout the specification.

Notwithstanding that the numerical ranges and parameters setting forththe broad scope of the disclosure are approximations, the numericalvalues set forth in the specific examples are reported as precisely aspossible. Any numerical value, however, inherently contains certainerrors necessarily resulting from the standard deviation found in theirrespective testing measurements. Moreover, all ranges disclosed hereinare to be understood to encompass any and all subranges subsumedtherein. For example, a stated range of “1 to 10” should be consideredto include any and all subranges between (and inclusive of) the minimumvalue of 1 and the maximum value of 10; that is, all subranges beginningwith a minimum value of 1 or more, e.g. 1 to 6.1, and ending with amaximum value of 10 or less, e.g., 5.5 to 10. Additionally, anyreference referred to as being “incorporated herein” is to be understoodas being incorporated in its entirety.

It is further noted that, as used in this specification, the singularforms “a,” “an,” and “the” include plural referents unless expressly andunequivocally limited to one referent. The term “and/or” generally isused to refer to at least one or the other. In some case the term“and/or” is used interchangeably with the term “or.” The term“including” is used herein to mean, and is used interchangeably with,the phrase “including but not limited to.” The term “such as” is usedherein to mean, and is used interchangeably with, the phrase “such asbut not limited to.”

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art. Practitioners are particularly directed to Current Protocols inMolecular Biology (Ausubel) for definitions and terms of the art.

Also as used herein, “at least one” contemplates any number from 1 tothe entire group. For example, for a listing of four markers, the phraseat “least one” is understood to mean 1, 2, 3 or 4 markers.

Also, as used herein, “comprising” includes embodiments moreparticularly defined using the term “consisting of.”

Antibody: As used herein, the term “antibody” refers to a polypeptideconsisting of one or more polypeptides substantially encoded byimmunoglobulin genes or fragments of immunoglobulin genes. Therecognized immunoglobulin genes include the kappa, lambda, alpha, gamma,delta, epsilon and mu constant region genes, as well as myriadimmunoglobulin variable region genes. Light chains are typicallyclassified as either kappa or lambda. Heavy chains are typicallyclassified as gamma, mu, alpha, delta, or epsilon, which in turn definethe immunoglobulin classes, IgG, IgM, IgA, IgD and IgE, respectively. Atypical immunoglobulin (antibody) structural unit is known to comprise atetramer. Each tetramer is composed of two identical pairs ofpolypeptide chains, each pair having one “light” (about 25 kD) and one“heavy” chain (about 50-70 kD). The N-terminus of each chain defines avariable region of about 100 to 110 or more amino acids primarilyresponsible for antigen recognition. The terms “variable light chain”(VL) and “variable heavy chain” (VH) refer to these light and heavychains respectively. An antibody can be specific for a particularantigen. The antibody or its antigen can be either an analyte or abinding partner. Antibodies exist as intact immunoglobulins or as anumber of well-characterized fragments produced by digestion withvarious peptidases. Thus, for example, pepsin digests an antibody belowthe disulfide linkages in the hinge region to produce F(ab)′2, a dimerof Fab which itself is a light chain joined to VH-CH1 by a disulfidebond. The F(ab)′2 may be reduced under mild conditions to break thedisulfide linkage in the hinge region thereby converting the (Fab′)2dimer into an Fab′ monomer. The Fab′ monomer is essentially an Fab withpart of the hinge region (see, Fundamental Immunology, W. E. Paul, ed.,Raven Press, N.Y. (1993), for a more detailed description of otherantibody fragments). While various antibody fragments are defined interms of the digestion of an intact antibody, one of ordinary skill inthe art will appreciate that such Fab′ fragments may be synthesized denovo either chemically or by utilizing recombinant DNA methodology.Thus, the term “antibody,” as used herein also includes antibodyfragments either produced by the modification of whole antibodies orsynthesized de novo using recombinant DNA methodologies. In someembodiments, antibodies are single chain antibodies, such as singlechain Fv (scFv) antibodies in which a variable heavy and a variablelight chain are joined together (directly or through a peptide linker)to form a continuous polypeptide. A single chain Fv (“scFv”) polypeptideis a covalently linked VH::VL heterodimer which may be expressed from anucleic acid including VH- and VL-encoding sequences either joineddirectly or joined by a peptide-encoding linker. (See, e.g., Huston, etal. (1988) Proc. Nat. Acad. Sci. USA, 85:5879-5883, the entire contentsof which are herein incorporated by reference.) A number of structuresexist for converting the naturally aggregated, but chemically separatedlight and heavy polypeptide chains from an antibody V region into anscFv molecule which will fold into a three dimensional structuresubstantially similar to the structure of an antigen-binding site. See,e.g. U.S. Pat. Nos. 5,091,513 and 5,132,405 and 4,956,778.

The term “antibody” includes monoclonal antibodies, polyclonalantibodies, synthetic antibodies and chimeric antibodies, e.g.,generated by combinatorial mutagenesis and phage display. The term“antibody” also includes mimetics or peptidomimetics of antibodies.Peptidomimetics are compounds based on, or derived from, peptides andproteins. The peptidomimetics of the present disclosure typically can beobtained by structural modification of a known peptide sequence usingunnatural amino acids, conformational restraints, isosteric replacement,and the like.

Allele: As used herein, the term “allele” refers to different versionsof a nucleotide sequence of a same genetic locus (e.g., a gene).

Allele specific primer extension (ASPE): As used herein, the term“allele specific primer extension (ASPE)” refers to a mutation detectionmethod utilizing primers which hybridize to a corresponding DNA sequenceand which are extended depending on the successful hybridization of the3′ terminal nucleotide of such primer. Typically, extension primers thatpossess a 3′ terminal nucleotide which form a perfect match with thetarget sequence are extended to form extension products. Modifiednucleotides can be incorporated into the extension product, suchnucleotides effectively labeling the extension products for detectionpurposes. Alternatively, an extension primer may instead comprise a 3′terminal nucleotide which forms a mismatch with the target sequence. Inthis instance, primer extension does not occur unless the polymeraseused for extension inadvertently possesses exonuclease activity.

Amplification: As used herein, the term “amplification” refers to anymethods known in the art for copying a target nucleic acid, therebyincreasing the number of copies of a selected nucleic acid sequence.Amplification may be exponential or linear. A target nucleic acid may beeither DNA or RNA. Typically, the sequences amplified in this mannerform an “amplicon.” Amplification may be accomplished with variousmethods including, but not limited to, the polymerase chain reaction(“PCR”), transcription-based amplification, isothermal amplification,rolling circle amplification, etc. Amplification may be performed withrelatively similar amount of each primer of a primer pair to generate adouble stranded amplicon. However, asymmetric PCR may be used to amplifypredominantly or exclusively a single stranded product as is well knownin the art (e.g., Poddar et al. Molec. And Cell. Probes 14:25-32(2000)). This can be achieved using each pair of primers by reducing theconcentration of one primer significantly relative to the other primerof the pair (e.g., 100 fold difference). Amplification by asymmetric PCRis generally linear. A skilled artisan will understand that differentamplification methods may be used together.

Animal: As used herein, the term “animal” refers to any member of theanimal kingdom. In some embodiments, “animal” refers to humans, at anystage of development. In some embodiments, “animal” refers to non-humananimals, at any stage of development. In certain embodiments, thenon-human animal is a mammal (e.g., a rodent, a mouse, a rat, a rabbit,a monkey, a dog, a cat, a sheep, cattle, a primate, and/or a pig). Insome embodiments, animals include, but are not limited to, mammals,birds, reptiles, amphibians, fish, insects, and/or worms. In someembodiments, an animal may be a transgenic animal,genetically-engineered animal, and/or a clone.

Approximately: As used herein, the term “approximately” or “about,” asapplied to one or more values of interest, refers to a value that issimilar to a stated reference value. In certain embodiments, the term“approximately” or “about” refers to a range of values that fall within25%, 20%, 19%, 18%, 17%, 16%, 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%,6%, 5%, 4%, 3%, 2%, 1%, or less in either direction (greater than orless than) of the stated reference value unless otherwise stated orotherwise evident from the context (except where such number wouldexceed 100% of a possible value). Also, throughout this application, theterm “about” is used to indicate that a value includes the inherentvariation of error for the device, the method being employed todetermine the value, or the variation that exists among samples.

Associated with a syndrome or disease of interest: As used herein,“associated with a syndrome or disease of interest” means that thevariant is found with in patients with the syndrome or disease ofinterest more than in non-syndromic or non-disease controls. Generally,the statistical significance of such association can be determined byassaying a plurality of patients.

Biological Sample and Sample: As used herein, the term “biologicalsample” encompasses any sample obtained from a biological source. Abiological sample can, by way of non-limiting example, include blood,serum, plasma, tissue biopsty, cell-free DNA, amniotic fluid, sera,urine, feces, epidermal sample, skin sample, cheek swab, sperm, amnioticfluid, cultured cells, bone marrow sample and/or chorionic villi.Convenient biological samples may be obtained by, for example, scrapingcells from the surface of the buccal cavity. The term biological sampleencompasses samples which have been processed to release or otherwisemake available a nucleic acid or protein for detection as describedherein. For example, a biological sample may include a cDNA that hasbeen obtained by reverse transcription of RNA from cells in a biologicalsample. The biological sample may be obtained from a stage of life suchas a fetus, young adult, adult, and the like. Fixed or frozen tissuesalso may be used.

Biomarker: As used herein, the term “biomarker” refers to one or morenucleic acids, polypeptides and/or other biomolecules (e.g.,cholesterol, lipids) that can be used to diagnose, or to aid in thediagnosis or prognosis of a disease or syndrome of interest, eitheralone or in combination with other biomarkers; monitor the progressionof a disease or syndrome of interest; and/or monitor the effectivenessof a treatment for a syndrome or a disease of interest.

Binding agent: As used herein, the term “binding agent” refers to amolecule that can specifically and selectively bind to a second (i.e.,different) molecule of interest. The interaction may be non-covalent,for example, as a result of hydrogen-bonding, van der Waalsinteractions, or electrostatic or hydrophobic interactions, or it may becovalent. The term “soluble binding agent” refers to a binding agentthat is not associated with (i.e., covalently or non-covalently bound)to a solid support.

Carrier: The term “carrier” refers to a person who is symptom-free butcarries a mutation that can be passed to his/her children. Typically,for an autosomal recessive disorder, a carrier has one allele thatcontains a disease causing mutation and a second allele that is normalor not disease-related.

Coding sequence vs. non-coding sequence: As used herein, the term“coding sequence” refers to a sequence of a nucleic acid or itscomplement, or a part thereof, that can be transcribed and/or translatedto produce the mRNA for and/or the polypeptide or a fragment thereof.Coding sequences include exons in a genomic DNA or immature primary RNAtranscripts, which are joined together by the cell's biochemicalmachinery to provide a mature mRNA. The anti-sense strand is thecomplement of such a nucleic acid, and the encoding sequence can bededuced therefrom. As used herein, the term “non-coding sequence” refersto a sequence of a nucleic acid or its complement, or a part thereof,that is not transcribed into amino acid in vivo, or where tRNA does notinteract to place or attempt to place an amino acid. Non-codingsequences include both intron sequences in genomic DNA or immatureprimary RNA transcripts, and gene-associated sequences such aspromoters, enhancers, silencers, etc.

Complement: As used herein, the terms “complement,” “complementary” and“complementarity,” refer to the pairing of nucleotide sequencesaccording to Watson/Crick pairing rules. For example, a sequence5′-GCGGTCCCA-3′ has the complementary sequence of 5′-TGGGACCGC-3′. Acomplement sequence can also be a sequence of RNA complementary to theDNA sequence. Certain bases not commonly found in natural nucleic acidsmay be included in the complementary nucleic acids including, but notlimited to, inosine, 7-deazaguanine, Locked Nucleic Acids (LNA), andPeptide Nucleic Acids (PNA). Complementary need not be perfect; stableduplexes may contain mismatched base pairs, degenerative, or unmatchedbases. Those skilled in the art of nucleic acid technology can determineduplex stability empirically considering a number of variablesincluding, for example, the length of the oligonucleotide, basecomposition and sequence of the oligonucleotide, ionic strength andincidence of mismatched base pairs.

Conserved: As used herein, the term “conserved residues” refers to aminoacids that are the same among a plurality of proteins having the samestructure and/or function. A region of conserved residues may beimportant for protein structure or function. Thus, contiguous conservedresidues as identified in a three-dimensional protein may be importantfor protein structure or function. To find conserved residues, orconserved regions of 3-D structure, a comparison of sequences for thesame or similar proteins from different species, or of individuals ofthe same species, may be made.

Control: As used herein, the term “control” has its art-understoodmeaning of being a standard against which results are compared.Typically, controls are used to augment integrity in experiments byisolating variables in order to make a conclusion about such variables.In some embodiments, a control is a reaction or assay that is performedsimultaneously with a test reaction or assay to provide a comparator. Inone experiment, the “test” (i.e., the variable being tested) is applied.In the second experiment, the “control,” the variable being tested isnot applied. In some embodiments, a control is a historical control(i.e., of a test or assay performed previously, or an amount or resultthat is previously known). In some embodiments, a control is orcomprises a printed or otherwise saved record. A control may be apositive control or a negative control.

A “control” or “predetermined standard” for a biomarker refers to thelevels of expression of the biomarker in healthy subjects or theexpression levels of said biomarker in non-diseased or non-syndromictissue from the same subject. The control or predetermined standardexpression levels or amounts of protein for a given biomarker can beestablished by prospective and/or retrospective statistical studiesusing only routine experimentation. Such predetermined standardexpression levels and/or protein levels (amounts) can be determined by aperson having ordinary skill in the art using well known methods.

Crude: As used herein, the term “crude,” when used in connection with abiological sample, refers to a sample which is in a substantiallyunrefined state. For example, a crude sample can be cell lysates orbiopsy tissue sample. A crude sample may exist in solution or as a drypreparation.

Deletion: As used herein, the term “deletion” encompasses a mutationthat removes one or more nucleotides from a naturally-occurring nucleicacid.

Disease or syndrome of interest: As used herein, a disease or syndromeof interest is NCGS. NCGS as used here includes non-coeliac wheatsensitivity (NCWS).

Detect: As used herein, the term “detect”, “detected” or “detecting”includes “measure,” “measured” or“measuring” and vice versa.

Detectable moiety: As used herein, the term “detectable moiety” or“detectable biomolecule” or “reporter” refers to a molecule that can bemeasured in a quantitative assay. For example, a detectable moiety maycomprise an enzyme that may be used to convert a substrate to a productthat can be measured (e.g., a visible product). Or, a detectable moietymay be a radioisotope that can be quantified. Or, a detectable moietymay be a fluorophore. Or, a detectable moiety may be a luminescentmolecule. Or, other detectable molecules may be used.

Epigenetic: As used herein, an epigenetic element can change geneexpression by a mechanism other than a change in the underlying DNAsequences. Such elements may include elements that regulateparamutation, imprinting, gene silencing, X chromosome inactivation,position effect, reprogramming, transvection, maternal effects, histonemodification, and heterochromatin.

Epitope: As used herein, the term “epitope” refers to a fragment orportion of a molecule or a molecule compound (e.g., a polypeptide or aprotein complex) that makes contact with a particular antibody orantibody like proteins.

Exon: As used herein an exon is a nucleic acid sequence that is found inmature or processed RNA after other portions of the RNA (e.g.,intervening regions known as introns) have been removed by RNA splicing.As such, exon sequences generally encode for proteins or portions ofproteins. An intron is the portion of the RNA that is removed fromsurrounding exon sequences by RNA splicing.

Expression and expressed RNA: As used herein expressed RNA is an RNAthat encodes for a protein or polypeptide (“coding RNA”), and any otherRNA that is transcribed but not translated (“non-coding RNA”). The term“expression” is used herein to mean the process by which a polypeptideis produced from DNA. The process involves the transcription of the geneinto mRNA and the translation of this mRNA into a polypeptide. Dependingon the context in which used, “expression” may refer to the productionof RNA, protein or both.

The measurement of an amount of a protein and/or the expression of abiomarker of the disclosure may be assessed by any of a wide variety ofwell-known methods for detecting expression of a transcribed molecule orits corresponding protein. Non-limiting examples of such methods includeimmunological methods for detection of secreted proteins, proteinpurification methods, protein function or activity assays, nucleic acidhybridization methods, nucleic acid reverse transcription methods, andnucleic acid amplification methods. In certain embodiments, expressionof a marker gene is assessed using an antibody (e.g. a radio-labeled,chromophore-labeled, fluorophore-labeled, or enzyme-labeled antibody),an antibody derivative (e.g. an antibody conjugated with a substrate orwith the protein or ligand of a protein-ligand pair {e.g.biotin-streptavidin}), or an antibody fragment (e.g. a single-chainantibody, an isolated antibody hypervariable domain, etc.) which bindsspecifically with a protein corresponding to the marker gene, such asthe protein encoded by the open reading frame corresponding to themarker gene or such a protein which has undergone all or a portion ofits normal post-translational modification. In certain embodiments, areagent may be directly or indirectly labeled with a detectablesubstance. The detectable substance may be, for example, selected, e.g.,from a group consisting of radioisotopes, fluorescent compounds,enzymes, and enzyme co-factor. Methods of labeling antibodies are wellknown in the art.

In another embodiment, expression of a marker gene is assessed bypreparing mRNA/cDNA (i.e. a transcribed polynucleotide) from cells in asample, and by hybridizing the mRNA/cDNA with a reference polynucleotidewhich is a complement of a polynucleotide comprising the marker gene,and fragments thereof. cDNA can, optionally, be amplified using any of avariety of polymerase chain reaction methods prior to hybridization withthe reference polynucleotide; preferably, it is not amplified.

Familial history: As used herein, the term “familial history” typicallyrefers to occurrence of events (e.g., disease related disorder ormutation carrier) relating to an individual's immediate family membersincluding parents and siblings. Family history may also includegrandparents and other relatives.

Flanking: As used herein, the term “flanking” is meant that a primerhybridizes to a target nucleic acid adjoining a region of interestsought to be amplified on the target. The skilled artisan willunderstand that preferred primers are pairs of primers that hybridize 3′from a region of interest, one on each strand of a target doublestranded DNA molecule, such that nucleotides may be add to the 3′ end ofthe primer by a suitable DNA polymerase. For example, primers that flankmutant sequences do not actually anneal to the mutant sequence butrather anneal to sequence that adjoins the mutant sequence. In somecases, primers that flank an exon are generally designed not to annealto the exon sequence but rather to anneal to sequence that adjoins theexon (e.g. intron sequence). However, in some cases, amplificationprimer may be designed to anneal to the exon sequence.

Gene: As used herein a gene is a unit of heredity. Generally, a gene isa portion of DNA that encodes a protein or a functional RNA. A gene is alocatable region of genomic sequence corresponding to a unit ofinheritance. A gene may be associated with regulatory regions,transcribed regions, and or other functional sequence regions. Genotype:As used herein, the term “genotype” refers to the genetic constitutionof an organism. More specifically, the term refers to the identity ofalleles present in an individual. “Genotyping” of an individual or a DNAsample refers to identifying the nature, in terms of nucleotide base, ofthe two alleles possessed by an individual at a known polymorphic site.

Gene regulatory element: As used herein a gene regulatory element orregulatory sequence is a segment of DNA where regulatory proteins, suchas transcription factors, bind to regulate gene expression. Suchregulatory regions are often upstream of the gene being regulated.

Healthy individual: As used herein, the term “healthy individual” or“control” refers to a subject has not been diagnosed with the syndromeand/or disease of interest.

Heterozygous: As used herein, the term “heterozygous” or “BET” refers toan individual possessing two different alleles of the same gene. As usedherein, the term “heterozygous” encompasses “compound heterozygous” or“compound heterozygous mutant.” As used herein, the term “compoundheterozygous” refers to an individual possessing two different alleles.As used herein, the term “compound heterozygous mutant” refers to anindividual possessing two different copies of an allele, such allelesare characterized as mutant forms of a gene.

Homozygous: As used herein, the term “homozygous” refers to anindividual possessing two copies of the same allele. As used herein, theterm “homozygous mutant” refers to an individual possessing two copiesof the same allele, such allele being characterized as the mutant formof a gene.

Hybridize: As used herein, the term “hybridize” or “hybridization”refers to a process where two complementary nucleic acid strands annealto each other under appropriately stringent conditions. Oligonucleotidesor probes suitable for hybridizations typically contain 10-100nucleotides in length (e.g., 18-50, 12-70, 10-30, 10-24, 18-36nucleotides in length). Nucleic acid hybridization techniques are wellknown in the art. Those skilled in the art understand how to estimateand adjust the stringency of hybridization conditions such thatsequences having at least a desired level of complementary will stablyhybridize, while those having lower complementary will not. For examplesof hybridization conditions and parameters, see, e.g., Sambrook, et al.,1989, Molecular Cloning: A Laboratory Manual, Second Edition, ColdSpring Harbor Press, Plainview, N.Y.; Ausubel, F. M. et al. 1994,Current Protocols in Molecular Biology. John Wiley & Sons, Secaucus,N.J.

Identity or percent identical: As used herein, the terms “identity” or“percent identical” refers to sequence identity between two amino acidsequences or between two nucleic acid sequences. Percent identity can bedetermined by aligning two sequences and refers to the number ofidentical residues (i.e., amino acid or nucleotide) at positions sharedby the compared sequences. Sequence alignment and comparison may beconducted using the algorithms standard in the art (e.g. Smith andWaterman, 1981, Adv. Appl. Math. 2:482; Needleman and Wunsch, 1970, J.Mol. Biol. 48:443; Pearson and Lipman, 1988, Proc. Natl. Acad. Sci.,USA, 85:2444) or by computerized versions of these algorithms (WisconsinGenetics Software Package Release 7.0, Genetics Computer Group, 575Science Drive, Madison, Wis.) publicly available as BLAST and FASTA.Also, ENTREZ, available through the National Institutes of Health,Bethesda Md., may be used for sequence comparison. In other cases,commercially available software, such as GenomeQuest, may be used todetermine percent identity. When utilizing BLAST and Gapped BLASTprograms, the default parameters of the respective programs (e.g.,BLASTN; available at the Internet site for the National Center forBiotechnology Information) may be used. In one embodiment, the percentidentity of two sequences may be determined using GCG with a gap weightof 1, such that each amino acid gap is weighted as if it were a singleamino acid mismatch between the two sequences. Or, the ALIGN program(version 2.0), which is part of the GCG (Accelrys, San Diego, Calif.)sequence alignment software package may be used.

As used herein, the term at least 90% identical thereto includessequences that range from 90 to 100% identity to the indicated sequencesand includes all ranges in between. Thus, the term at least 90%identical thereto includes sequences that are 91, 91.5, 92, 92.5, 93,93.5, 94, 94.5, 95, 95.5, 96, 96.5, 97, 97.5, 98, 98.5, 99, 99.5 percentidentical to the indicated sequence. Similarly, the term “at least 70%identical includes sequences that range from 70 to 100% identical, withall ranges in between. The determination of percent identity isdetermined using the algorithms described herein.

Insertion or addition: As used herein, the term “insertion” or“addition” refers to a change in an amino acid or nucleotide sequenceresulting in the addition of one or more amino acid residues ornucleotides, respectively, as compared to the naturally occurringmolecule.

In vitro: As used herein, the term “in vitro” refers to events thatoccur in an artificial environment, e.g., in a test tube or reactionvessel, in cell culture, etc., rather than within a multi-cellularorganism.

In vivo: As used herein, the term “in vivo” refers to events that occurwithin a multi-cellular organism such as a non-human animal.

Isolated: As used herein, the term “isolated” refers to a substanceand/or entity that has been (1) separated from at least some of thecomponents with which it was associated when initially produced (whetherin nature and/or in an experimental setting), and/or (2) produced,prepared, and/or manufactured by the hand of man. Isolated substancesand/or entities may be separated from at least about 10%, about 20%,about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about90%, about 95%, about 98%, about 99%, substantially 100%, or 100% of theother components with which they were initially associated. In someembodiments, isolated agents are more than about 80%, about 85%, about90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%,about 97%, about 98%, about 99%, substantially 100%, or 100% pure. Asused herein, a substance is “pure” if it is substantially free of othercomponents. As used herein, the term “isolated cell” refers to a cellnot contained in a multi-cellular organism.

Labeled: The terms “labeled” and “labeled with a detectable agent ormoiety” are used herein interchangeably to specify that an entity (e.g.,a nucleic acid probe, antibody, etc.) can be measured by detection ofthe label (e.g., visualized, detection of radioactivity and the like)for example following binding to another entity (e.g., a nucleic acid,polypeptide, etc.). The detectable agent or moiety may be selected suchthat it generates a signal which can be measured and whose intensity isrelated to (e.g., proportional to) the amount of bound entity. A widevariety of systems for labeling and/or detecting proteins and peptidesare known in the art. Labeled proteins and peptides can be prepared byincorporation of, or conjugation to, a label that is detectable byspectroscopic, photochemical, biochemical, immunochemical, electrical,optical, chemical or other means. A label or labeling moiety may bedirectly detectable (i.e., it does not require any further reaction ormanipulation to be detectable, e.g., a fluorophore is directlydetectable) or it may be indirectly detectable (i.e., it is madedetectable through reaction or binding with another entity that isdetectable, e.g., a hapten is detectable by immunostaining afterreaction with an appropriate antibody comprising a reporter such as afluorophore). Suitable detectable agents include, but are not limitedto, radionucleotides, fluorophores, chemiluminescent agents,microparticles, enzymes, colorimetric labels, magnetic labels, haptens,molecular beacons, aptamer beacons, and the like.

Micro RNA: As used herein microRNAs (miRNAs) are short (20-24nucleotide) non-coding RNAs that are involved in post-transcriptionalregulation of gene expression. microRNA can affect both the stabilityand translation of mRNAs. For example, microRNAs can bind tocomplementary sequences in the 3′UTR of target mRNAs and cause genesilencing. miRNAs are transcribed by RNA polymerase II as part of cappedand polyadenylated primary transcripts (pri-miRNAs) that can be eitherprotein-coding or non-coding. The primary transcript can be cleaved bythe Drosha ribonuclease III enzyme to produce an approximately70-nucleotide stem-loop precursor miRNA (pre-miRNA), which can furtherbe cleaved by the cytoplasmic Dicer ribonuclease to generate the maturemiRNA and antisense miRNA star (miRNA*) products. The mature miRNA canbe incorporated into a RNA-induced silencing complex (RISC), which canrecognize target mRNAs through imperfect base pairing with the miRNA andmost commonly results in translational inhibition or destabilization ofthe target mRNA.

Multiplex PCR: As used herein, the term “multiplex PCR” refers toconcurrent amplification of two or more regions which are each primedusing a distinct primers pair.

Multiplex ASPE: As used herein, the term “multiplex ASPE” refers to anassay combining multiplex PCR and allele specific primer extension(ASPE) for detecting polymorphisms. Typically, multiplex PCR is used tofirst amplify regions of DNA that will serve as target sequences forASPE primers. See the definition of allele specific primer extension.

Mutation and/or variant: As used herein, the terms mutation and variantare used interchangeably to describe a nucleic acid or protein sequencechange. The term “mutant” as used herein refers to a mutated, orpotentially non-functional form of a gene. The term includes anymutation that renders a gene not functional from a point mutation tolarge chromosomal rearrangements as is known in the art.

Nucleic acid. As used herein, a “nucleic acid” is a polynucleotide suchas deoxyribonucleic acid (DNA) or ribonucleic acid (RNA). The term isused to include single-stranded nucleic acids, double-stranded nucleicacids, and RNA and DNA made from nucleotide or nucleoside analogues.

Polypeptide or protein: As used herein, the term “polypeptide” and/or“protein” refers to a polymer of amino acids, and not to a specificlength. Thus, peptides, oligopeptides and proteins are included withinthe definition of polypeptide and/or protein. “Polypeptide” and“protein” are used interchangeably herein to describe protein moleculesthat may comprise either partial or full-length proteins. The term“peptide” is used to denote a less than full-length protein or a veryshort protein unless the context indicates otherwise.

As is known in the art, “proteins”, “peptides,” “polypeptides” and“oligopeptides” are chains of amino acids (typically L-amino acids)whose alpha carbons are linked through peptide bonds formed by acondensation reaction between the carboxyl group of the alpha carbon ofone amino acid and the amino group of the alpha carbon of another aminoacid. Typically, the amino acids making up a protein are numbered inorder, starting at the amino terminal residue and increasing in thedirection toward the carboxy terminal residue of the protein.Abbreviations for amino acid residues are the standard 3-letter and/or1-letter codes used in the art to refer to one of the 20 common L-aminoacids.

As used herein, a polypeptide or protein “domain” comprises a regionalong a polypeptide or protein that comprises an independent unit.Domains may be defined in terms of structure, sequence and/or biologicalactivity. In one embodiment, a polypeptide domain may comprise a regionof a protein that folds in a manner that is substantially independentfrom the rest of the protein. Domains may be identified using domaindatabases such as, but not limited to PFAM, PRODOM, PROSITE, BLOCKS,PRINTS, SBASE, ISREC PROFILES, SAMRT, and PROCLASS.

Primer: As used herein, the term “primer” refers to a shortsingle-stranded oligonucleotide capable of hybridizing to acomplementary sequence in a nucleic acid sample. Typically, a primerserves as an initiation point for template dependent DNA synthesis.Deoxyribonucleotides can be added to a primer by a DNA polymerase. Insome embodiments, such deoxyribonucleotides addition to a primer is alsoknown as primer extension. The term primer, as used herein, includes allforms of primers that may be synthesized including peptide nucleic acidprimers, locked nucleic acid primers, phosphorothioate modified primers,labeled primers, and the like. A “primer pair” or “primer set” for a PCRreaction typically refers to a set of primers typically including a“forward primer” and a “reverse primer.” As used herein, a “forwardprimer” refers to a primer that anneals to the anti-sense strand ofdsDNA. A “reverse primer” anneals to the sense-strand of dsDNA.

Polymorphism: As used herein, the term “polymorphism” refers to thecoexistence of more than one form of a gene or portion thereof.

Portion and Fragment: As used herein, the terms “portion” and “fragment”are used interchangeably to refer to parts of a polypeptide, nucleicacid, or other molecular construct.

Sense strand vs. anti-sense strand: As used herein, the term “sensestrand” refers to the strand of double-stranded DNA (dsDNA) thatincludes at least a portion of a coding sequence of a functionalprotein. As used herein, the term “anti-sense strand” refers to thestrand of dsDNA that is the reverse complement of the sense strand.

Significant difference: As used herein, the term “significantdifference” is well within the knowledge of a skilled artisan and willbe determined empirically with reference to each particular biomarker.For example, a significant difference in the expression of a biomarkerin a subject with the disease or syndrome of interest as compared to ahealthy subject is any difference in protein amounts which isstatistically significant.

Similar or homologue: As used herein, the term “similar” or “homologue”when referring to amino acid or nucleotide sequences means a polypeptidehaving a degree of homology or identity with the wild-type amino acidsequence. Homology comparisons can be conducted by eye, or more usually,with the aid of readily available sequence comparison programs. Thesecommercially available computer programs can calculate percent homologybetween two or more sequences (e.g. Wilbur, W. J. and Lipman, D. J.,1983, Proc. Natl. Acad. Sci. USA, 80:726-730). For example, homologoussequences may be taken to include an amino acid sequences which inalternate embodiments are at least 70% identical, 75% identical, 80%identical, 85% identical, 90% identical, 95% identical, 97% identical,or 98% identical to each other.

Specific: As used herein, the term “specific,” when used in connectionwith an oligonucleotide primer, refers to an oligonucleotide or primer,which under appropriate hybridization or washing conditions, is capableof hybridizing to the target of interest and not substantiallyhybridizing to nucleic acids which are not of interest. Higher levels ofsequence identity are preferred and include at least 60%, 65%, 70%, 75%,80%, 85%, 90%, 95%, 98%, 99%, or 100% sequence identity. In someembodiments, a specific oligonucleotide or primer contains at least 4,6, 8, 10, 12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 35, 40, 45, 50, 55,60, 65, 70, or more bases of sequence identity with a portion of thenucleic acid to be hybridized or amplified when the oligonucleotide andthe nucleic acid are aligned.

As is known in the art, conditions for hybridizing nucleic acidsequences to each other can be described as ranging from low to highstringency. Generally, highly stringent hybridization conditions referto washing hybrids in low salt buffer at high temperatures.Hybridization may be to filter bound DNA using hybridization solutionsstandard in the art such as 0.5 M NaHPO₄, 7% sodium dodecyl sulfate(SDS), at 65° C., and washing in 0.25 M NaHPO₄, 3.5% SDS followed bywashing 0.1×SSC/0.1% SDS at a temperature ranging from room temperatureto 68° C. depending on the length of the probe (see e.g. Ausubel, F. M.et al., Short Protocols in Molecular Biology, 4^(th), Ed., Chapter 2,John Wiley & Sons, N.Y). For example, a high stringency wash compriseswashing in 6×SSC/0.05% sodium pyrophosphate at 37° C. for a 14 baseoligonucleotide probe, or at 48° C. for a 17 base oligonucleotide probe,or at 55° C. for a 20 base oligonucleotide probe, or at 60° C. for a 25base oligonucleotide probe, or at 65° C. for a nucleotide probe about250 nucleotides in length. Nucleic acid probes may be labeled withradionucleotides by end-labeling with, for example, [γ-³²P]ATP, orincorporation of radiolabeled nucleotides such as [α-³²P]dCTP by randomprimer labeling. Alternatively, probes may be labeled by incorporationof biotinylated or fluorescein labeled nucleotides, and the probedetected using streptavidin or anti-fluorescein antibodies.

siRNA: As used herein, siRNA (small inhibitory RNA) is essentially adouble-stranded RNA molecule composed of about 20 complementarynucleotides. siRNA is created by the breakdown of larger double-stranded(ds) RNA molecules. siRNA can suppress gene expression by inherentlysplitting its corresponding mRNA in two by way of the interaction of thesiRNA with the mRNA, leading to degradation of the mRNA. siRNAs can alsointeract with DNA to facilitate chromatin silencing and the expansion ofheterochromatin.

Subject: As used herein, the term “subject” refers to a human or anynon-human animal. A subject can be a patient, which refers to a humanpresenting to a medical provider for diagnosis or treatment of adisease. A human includes pre and post-natal forms. Also, as usedherein, the terms “individual,” “subject” or “patient” includes allwarm-blooded animals. In one embodiment the subject is a human. In oneembodiment, the individual is a subject who has NCGS or has an enhancedrisk of developing NCGS.

Substantially: As used herein, the term “substantially” refers to thequalitative condition of exhibiting total or near-total extent or degreeof a characteristic or property of interest. One of ordinary skill inthe biological arts will understand that biological and chemicalphenomena rarely, if ever, go to completion and/or proceed tocompleteness or achieve or avoid an absolute result. The term“substantially” is therefore used herein to capture the potential lackof completeness inherent in many biological and chemical phenomena.

Substantially complementary: As used herein, the term “substantiallycomplementary” refers to two sequences that can hybridize understringent hybridization conditions. The skilled artisan will understandthat substantially complementary sequences need not hybridize alongtheir entire length. In some embodiments, “stringent hybridizationconditions” refer to hybridization conditions at least as stringent asthe following: hybridization in 50% formamide, 5×SSC, 50 mM NaH₂PO₄, pH6.8, 0.5% SDS, 0.1 mg/mL sonicated salmon sperm DNA, and 5×Denhart'ssolution at 42° C. overnight; washing with 2×SSC, 0.1% SDS at 45° C.;and washing with 0.2×SSC, 0.1% SDS at 45° C. In some embodiments,stringent hybridization conditions should not allow for hybridization oftwo nucleic acids which differ over a stretch of 20 contiguousnucleotides by more than two bases.

Substitution: As used herein, the term “substitution” refers to thereplacement of one or more amino acids or nucleotides by different aminoacids or nucleotides, respectively, as compared to the naturallyoccurring molecule.

Suffering from: An individual who is “suffering from” a disease,disorder, and/or condition has been diagnosed with or displays one ormore symptoms of the disease, disorder, and/or condition.

Susceptible to: An individual who is “susceptible to” a disease,disorder, and/or condition has not been diagnosed with the disease,disorder, and/or condition. In some embodiments, an individual who issusceptible to a disease, disorder, and/or condition may not exhibitsymptoms of the disease, disorder, and/or condition. In someembodiments, an individual who is susceptible to a disease, disorder,and/or condition will develop the disease, disorder, and/or condition.In some embodiments, an individual who is susceptible to a disease,disorder, and/or condition will not develop the disease, disorder,and/or condition.

Solid support: The term “solid support” or “support” means a structurethat provides a substrate onto which biomolecules may be bound. Forexample, a solid support may be an assay well (i.e., such as amicrotiter plate), or the solid support may be a location on an array,or a mobile support, such as a bead.

Upstream and downstream: As used herein, the term “upstream” refers to aresidue that is N-terminal to a second residue where the molecule is aprotein, or 5′ to a second residue where the molecule is a nucleic acid.Also as used herein, the term “downstream” refers to a residue that isC-terminal to a second residue where the molecule is a protein, or 3′ toa second residue where the molecule is a nucleic acid. Protein,polypeptide and peptide sequences disclosed herein are all listed fromN-terminal amino acid to C-terminal acid and nucleic acid sequencesdisclosed herein are all listed from the 5′ end of the molecule to the3′ end of the molecule.

Overview

The disclosure herein provides novel mutations identified in a diseaseand/or syndrome of interest gene that can be used for more accuratediagnosis of disorders relating to the gene and/or syndrome of interest.

In some embodiments, the sample contains nucleic acid. In someembodiments, the testing step comprises nucleic acid sequencing. In someembodiments, the testing step comprises hybridization. In someembodiments, the hybridization is performed using one or moreoligonucleotide probes specific for a region in the biomarker ofinterest. In some embodiments, for detection of mutations, hybridizationis performed under conditions sufficiently stringent to disallow asingle nucleotide mismatch. In some embodiments, the hybridization isperformed with a microarray. In some embodiments, the testing stepcomprises restriction enzyme digestion. In some embodiments, the testingstep comprises PCR amplification. In some embodiments, the PCRamplification is digital PCR amplification. In some embodiments, thetesting step comprises primer extension. In some embodiments, the primerextension is single-base primer extension. In some embodiments, thetesting step comprises performing a multiplex allele-specific primerextension (ASPE).

In some embodiments, the sample contains protein. In some embodiments,the testing step comprises amino acid sequencing. In some embodiments,the testing step comprises performing an immunoassay using one or moreantibodies that specifically recognize the biomarker of interest. Insome embodiments, the testing step comprises protease digestion (e.g.,trypsin digestion). In some embodiments, the testing step furthercomprises performing 2D-gel electrophoresis.

In some embodiments, the testing step comprises determining the presenceof the one or more biomarkers using mass spectrometry. In someembodiments, the mass spectrometric format is selected from amongMatrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF),Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), FourierTransform, and combinations thereof.

In some embodiments, the sample is obtained from cells, tissue, wholeblood, mouthwash, plasma, serum, urine, stool, saliva, cord blood,chorionic villus sample, chorionic villus sample culture, amnioticfluid, amniotic fluid culture, transcervical lavage fluid, orcombination thereof. In further embodiments, the sample is obtained fromblood or blood products (e.g., plasma or serum) from a pregnant womanand/or fetal DNA.

In some embodiments, the testing step comprises determining the identityof the nucleotide and/or amino acid at a pre-determined position in thebiomarker. In some embodiments, the presence of the mutation isdetermined by comparing the identity of the nucleotide and/or amino acidat the pre-determined position to a control.

In embodiments, the method may comprise performing the assay (e.g.,sequencing) in a plurality of individuals to determine the statisticalsignificance of the association.

In another aspect, the disclosure provides reagents for detecting thebiomarker of interest such as, but not limited to a nucleic acid probethat specifically binds to the biomarker (e.g., a mutation in a DNAsequence), or an array containing one or more probes that specificallybind to the biomarker. In some embodiments, the disclosure provides anantibody that specifically binds to the biomarker. In some embodiments,the disclosure provides a kit for comprising one or more of suchreagents. In some embodiments, the one or more reagents are provided ina form of microarray. In some embodiments, the kit further comprisesreagents for primer extension. In some embodiments, the kit furthercomprises a control indicative of a healthy individual. In someembodiments, the kit further comprises an instructions on how todetermine if an individual has the syndrome or disease of interest basedon the biomarker of interest.

In some cases, the amount of the one or more biomarkers may, in certainembodiments, be detected by: (a) detecting the amount of a polypeptideor protein which is regulated by said one or more biomarker; (b)detecting the amount of a polypeptide or protein which regulates saidbiomarker; or (c) detecting the amount of a metabolite of the biomarker.

In still another aspect, the disclosure herein provides a computerreadable medium encoding information corresponding detection of thebiomarker.

Methods and Compositions for Diagnosing NCGS

Embodiments of the present disclosure comprise compositions and methodsfor diagnosing presence or increased risk of developing non-coeliacgluten sensitivity (NCGS). The methods and compositions of the presentdisclosure may be used to obtain or provide genetic information from asubject in order to objectively diagnose the presence or increased riskfor that subject, or other subjects to develop NCGS. The methods andcompositions may be embodied in a variety of ways.

In one embodiment, disclosed is a method to detect a biomarkerassociated with non-coeliac gluten sensitivity (NCGS) in an individualcomprising the steps of: obtaining a sample from the individual; andmeasuring the amount of at least one of IL-8, IL-10, TNF-Alpha or totalIgE protein in the sample. In some cases, the measuring may furthercomprise measuring expression of one of IP-10, CD4 or CD45. Additionallyand/or alternatively, the method may include measurement of at least onenormalization (e.g., housekeeping) gene. In one non-limiting embodiment,the housekeeping gene may be glyceraldehyde 3-phosphate dehydrogenase.Or, other house keeping genes may be used. Additionally and/oralternatively, other biomarkers may be measured.

As disclosed herein, a variety of methods may be used to measure thebiomarkers of interest. In one embodiment, the measuring comprisesmeasuring peptide or polypeptide biomarkers. For example, in oneembodiment, the measuring comprises an immunoassay. Or, the measuringmay comprise flow cytometry. Or, as discussed in detail herein, nucleicacid methods may be used.

A variety of sample types may be used. In certain embodiments, thesample comprises blood, serum, plasma, or a tissue biopsy.

In certain embodiments, the disclosure provides a method of identifyinga marker associated with NCGS in an individual. The method may comprisethe steps of identifying at least one marker having increased ordecreased expression in NCGS, but not in coeliac disease as compared toa control individual or population.

In other embodiments, the disclosure provides a method to detect thepresence of, or susceptibility to, non-coeliac gluten sensitivity (NCGS)in an individual. The method may comprise the steps of obtaining asample from the individual; measuring the amount of at least one ofIL-8, IL-10, TNF-Alpha or total IgE protein in the sample; and comparingthe expression of the at least one of IL-8, IL-10, TNF-alpha or totalIgE in the sample with a control value for each of the IL-8, IL-10,TNF-alpha or total IgE. In some cases, the measuring may furthercomprise measuring expression of at least one of IP-10, CD4 or CD45 andcomparing the levels of these markers with that of a control value. Inan embodiment, the control value is derived from is a healthy individualor individuals with no detected or detectable gastrointestinalpathology. In some embodiments, the control is a disease control. Suchdisease controls may include individuals with coeliac disease(stratified by whether the individual is on a gluten-free diet or not ona gluten free diet) and subjects with other GI diseases such asinflammatory bowel disease (IBD), hepatitis, small intestinal bacterialovergrowth, and other diseases.

Additionally and/or alternatively, the method may include measurement ofat least one normalization (e.g., housekeeping) gene. In onenon-limiting embodiment, the housekeeping gene may be glyceraldehyde3-phosphate dehydrogenase. Or, other housekeeping genes may be used.Additionally and/or alternatively, other biomarkers may be measured.

As disclosed herein, a variety of methods may be used to measure thebiomarkers of interest. In one embodiment, the measuring comprisesmeasuring peptide or polypeptide biomarkers. For example, in oneembodiment, the measuring comprises an immunoassay. Or, the measuringmay comprise flow cytometry. Or, as discussed in detail herein, nucleicacid methods may be used.

A variety of sample types may be used. In certain embodiments, thesample comprises blood, serum, plasma or a tissue biopsy.

Yet other embodiments comprise a composition to detect biomarkersassociated with non-coeliac gluten sensitivity (NCGS) in an individual.In certain embodiments, the composition comprises reagents that quantifythe levels of at least one of IL-8, IL-10, TNF-alpha or total IgEprotein in a biological sample. In some cases, the composition mayfurther comprise reagents for measuring expression of at least one ofIP-10, CD4 or CD45.

For example, as described in detail herein the composition may comprisereagents to measure peptide or polypeptide biomarkers. In oneembodiment, the composition comprises reagents to perform animmunoassay. Or, the composition may comprise reagents to perform flowcytometry. Or, as discussed in detail herein, the composition maycomprise reagents to determine the presence of a particular sequenceand/or expression level of a nucleic acid.

Other embodiments include kits that contain at least some of thecompositions disclosed herein and/or reagents for performing the methodsdisclosed herein. Such kits may include control biological samples fromis a healthy individual or individuals with no detected or detectablegastrointestinal pathology. In some embodiments, the control is adisease control. Such disease controls may include individuals withcoeliac disease (stratified by whether the individual is on agluten-free diet or not on a gluten free diet) and subjects with otherGI diseases such as inflammatory bowel disease (IBD), hepatitis, smallintestinal bacterial overgrowth, and other diseases. Such kits mayinclude instructions and/or computer-readable media comprisinginstructions and/or other information for performing the methods. Suchinstructions may comprise control values as described herein.

Other embodiments comprise computer-readable media comprisinginstructions and/or other information for performing the methodsindependent of a kit or reagents therein.

Peptide, Polypeptide and Protein Assays

In certain embodiments, the biomarker of interest is detected at theprotein (or peptide or polypeptide level), that is, a gene product isanalyzed. For example, a protein or fragment thereof can be analyzed byamino acid sequencing methods, or immunoassays using one or moreantibodies that specifically recognize one or more epitopes present onthe biomarker of interest, or in some cases specific to a mutation ofinterest. Proteins can also be analyzed by protease digestion (e.g.,trypsin digestion) and, in some embodiments, the digested proteinproducts can be further analyzed by 2D-gel electrophoresis.

Antibody Detection

Specific antibodies that bind the biomarker of interest can be employedin any of a variety of methods known in the art. Antibodies againstparticular epitopes, polypeptides, and/or proteins can be generatedusing any of a variety of known methods in the art. For example, theepitope, polypeptide, or protein against which an antibody is desiredcan be produced and injected into an animal, typically a mammal (such asa donkey, mouse, rabbit, horse, chicken, etc.), and antibodies producedby the animal can be collected from the animal. Monoclonal antibodiescan also be produced by generating hybridomas that express an antibodyof interest with an immortal cell line.

In some embodiments, antibodies are labeled with a detectable moiety asdescribed herein.

Antibody detection methods are well known in the art including, but arenot limited to, enzyme-linked immunoadsorbent assays (ELISAs) andWestern blots. Some such methods are amenable to being performed in anarray format.

For example, in some embodiments, the biomarker of interest is detectedusing a first antibody (or antibody fragment) that specificallyrecognizes the biomarker. The antibody may be labeled with a detectablemoiety (e.g., a chemiluminescent molecule), an enzyme, or a secondbinding agent (e.g., streptavidin). Or, the first antibody may bedetected using a second antibody, as is known in the art.

In certain embodiments, the method may further comprise adding a capturesupport, the capture support comprising at least one capture supportbinding agent that recognizes and binds to the biomarker so as toimmobilize the biomarker on the capture support. The method may, incertain embodiments, further comprise adding a second binding agent thatcan specifically recognize and bind to at least some of the pluralitybinding agent molecules on the capture support. In an embodiment, thebinding agent that can specifically recognize and bind to at least someof the plurality binding agent molecules on the capture support is asoluble binding agent (e.g., a secondary antibody). The second bindingagent may be labeled (e.g., with an enzyme) such that binding of thebiomarker of interest is measured by adding a substrate for the enzymeand quantifying the amount of product formed.

In an embodiment, the capture solid support may be an assay well (i.e.,such as a microtiter plate). Or, the capture solid support may be alocation on an array, or a mobile support, such as a bead. Or thecapture support may be a filter.

In some cases, the biomarker may be allowed to complex with a firstbinding agent (e.g., primary antibody specific for the biomarker andlabeled with detectable moiety) and a second binding agent (e.g., asecondary antibody that recognizes the primary antibody or a secondprimary antibody), where the second binding agent is complexed to athird binding agent (e.g., biotin) that can then interact with a capturesupport (e.g., magnetic bead) having a reagent (e.g., streptavidin) thatrecognizes the third binding agent linked to the capture support. Thecomplex (labeled primary antibody: biomarker: second primaryantibody-biotin: streptavidin-bead may then be captured using a magnet(e.g., a magnetic probe) to measure the amount of the complex.

A variety of binding agents may be used in the methods of thedisclosure. For example, the binding agent attached to the capturesupport, or the second antibody, may be either an antibody or anantibody fragment that recognizes the biomarker. Or, the binding agentmay comprise a protein that binds a non-protein target (i.e., such as aprotein that specifically binds to a small molecule biomarker, or areceptor that binds to a protein).

In certain embodiments, the solid supports may be treated with apassivating agent. For example, in certain embodiments the biomarker ofinterest may be captured on a passivated surface (i.e., a surface thathas been treated to reduce non-specific binding). One such passivatingagent is BSA. Additionally and/or alternatively, where the binding agentused is an antibody, the solid supports may be coated with protein A,protein G, protein A/G, protein L, or another agent that binds with highaffinity to the binding agent (e.g., antibody). These proteins bind theFc domain of antibodies and thus can orient the binding of antibodiesthat recognize the protein or proteins of interest.

Nucleic Acid Assays

In certain embodiments, the biomarkers disclosed herein are detected atthe nucleic acid level. In one embodiment, the disclosure comprisesmethods for diagnosing the presence or an increased risk of developingthe syndrome or disease of interest (e.g., NCGS) in a subject. Themethod may comprise the steps of obtaining a nucleic acid from a tissueor body fluid sample from a subject and conducting an assay to identifywhether there is a variant sequence (i.e., a mutation) in the subject'snucleic acid. In certain embodiments, the method may comprise comparingthe variant to known variants associated with the syndrome or disease ofinterest and determining whether the variant is a variant that has beenpreviously identified as being associated with the syndrome or diseaseof interest. Or, the method may comprise identifying the variant as anew, previously uncharacterized variant. If the variant is a newvariant, the method may further comprise performing an analysis todetermine whether the mutation is expected to be deleterious toexpression of the gene and/or the function of the protein encoded by thegene. The method may further comprise using the variant profile (i.e.,the compilation of mutations identified in the subject) to diagnose thepresence of the syndrome or disease of interest or an increased risk ofdeveloping the syndrome or disease of interest.

Nucleic acid analyses can be performed on genomic DNA, messenger RNAs,and/or cDNA. Also, in various embodiments, the nucleic acid comprises agene, an RNA, an exon, an intron, a gene regulatory element, anexpressed RNA, an siRNA, or an epigenetic element. Also, regulatoryelements, including splice sites, transcription factor binding, A-Iediting sites, microRNA binding sites, and functional RNA structuresites may be evaluated for mutations (i.e., variants). Thus, for each ofthe methods and compositions of the disclosure, the variant may comprisea nucleic acid sequence that encompasses at least one of the following:(1) A-to-I editing sites; (2) splice sites; (3) conserved functional RNAstructures; (4) validated transcription factor binding sites (TFBS); (5)microRNA (miRNA) binding sites; (6) polyadenylation sites; (7) knownregulatory elements; (8) miRNA genes; (9) small nucleolar RNA genesencoded in the ROIs; and/or (10) ultraconserved elements acrossplacental mammals.

In many embodiments, nucleic acids are extracted from a biologicalsample. In some embodiments, the nucleic acid is cell-free DNA. In someembodiments, nucleic acids are analyzed without having been amplified.In some embodiments, nucleic acids are amplified using techniques knownin the art (such as polymerase chain reaction (PCR)) and amplifiednucleic acids are used in subsequent analyses. Multiplex PCR, in whichseveral amplicons (e.g., from different genomic regions) are amplifiedat once using multiple sets of primer pairs, may be employed. Forexample, nucleic acid can be analyzed by sequencing, hybridization, PCRamplification, restriction enzyme digestion, primer extension such assingle-base primer extension or multiplex allele-specific primerextension (ASPE), or DNA sequencing. In some embodiments, nucleic acidsare amplified in a manner such that the amplification product for awild-type allele differs in size from that of a mutant allele. Thus,presence or absence of a particular mutant allele can be determined bydetecting size differences in the amplification products, e.g., on anelectrophoretic gel. For example, deletions or insertions of generegions may be particularly amenable to using size-based approaches.

Certain exemplary nucleic acid analysis methods are described in detailbelow.

Allele-Specific Amplification

In some embodiments, for example, where the biomarker for the diseaseand/or syndrome of interest is a mutation, a biomarker is detected usingan allele-specific amplification assay. This approach is variouslyreferred to as PCR amplification of specific allele (PASA) (Sarkar, etal., 1990 Anal. Biochem. 186:64-68), allele-specific amplification (ASA)(Okayama, et al., 1989 J. Lab. Clin. Med. 114:105-113), allele-specificPCR (ASPCR) (Wu, et al. 1989 Proc. Natl. Acad. Sci. USA. 86:2757-2760),and amplification-refractory mutation system (ARMS) (Newton, et al.,1989 Nucleic Acids Res. 17:2503-2516). The entire contents of each ofthese references is incorporated herein. This method is applicable forsingle base substitutions as well as micro deletions/insertions.

For example, for PCR-based amplification methods, amplification primersmay be designed such that they can distinguish between different alleles(e.g., between a wild-type allele and a mutant allele). Thus, thepresence or absence of amplification product can be used to determinewhether a gene mutation is present in a given nucleic acid sample. Insome embodiments, allele specific primers can be designed such that thepresence of amplification product is indicative of the gene mutation. Insome embodiments, allele specific primers can be designed such that theabsence of amplification product is indicative of the gene mutation.

In some embodiments, two complementary reactions are used. One reactionemploys a primer specific for the wild type allele (“wild-type-specificreaction”) and the other reaction employs a primer for the mutant allele(“mutant-specific reaction”). The two reactions may employ a commonsecond primer. PCR primers specific for a particular allele (e.g., thewild-type allele or mutant allele) generally perfectly match one allelicvariant of the target, but are mismatched to other allelic variant(e.g., the mutant allele or wild-type allele). The mismatch may belocated at/near the 3′ end of the primer, leading to preferentialamplification of the perfectly matched allele. Whether an amplificationproduct can be detected from one or in both reactions indicates theabsence or presence of the mutant allele. Detection of an amplificationproduct only from the wild-type-specific reaction indicates presence ofthe wild-type allele only (e.g., homozygosity of the wild-type allele).Detection of an amplification product in the mutant-specific reactiononly indicates presence of the mutant allele only (e.g. homozygosity ofthe mutant allele). Detection of amplification products from bothreactions indicate (e.g., a heterozygote). As used herein, this approachwill be referred to as “allele specific amplification (ASA).”

Allele-specific amplification can also be used to detect duplications,insertions, or inversions by using a primer that hybridizes partiallyacross the junction. The extent of junction overlap can be varied toallow specific amplification.

Amplification products can be examined by methods known in the art,including by visualizing (e.g., with one or more dyes) bands of nucleicacids that have been migrated (e.g., by electrophoresis) through a gelto separate nucleic acids by size.

Allele-Specific Primer Extension

In some embodiments, an allele-specific primer extension (ASPE) approachis used to detect a gene mutations. ASPE employs allele-specific primersthat can distinguish between alleles (e.g., between a mutant allele anda wild-type allele) in an extension reaction such that an extensionproduct is obtained only in the presence of a particular allele (e.g.,mutant allele or wild-type allele). Extension products may be detectableor made detectable, e.g., by employing a labeled deoxynucleotide in theextension reaction. Any of a variety of labels are compatible for use inthese methods, including, but not limited to, radioactive labels,fluorescent labels, chemiluminescent labels, enzymatic labels, etc. Insome embodiments, a nucleotide is labeled with an entity that can thenbe bound (directly or indirectly) by a detectable label, e.g., a biotinmolecule that can be bound by streptavidin-conjugated fluorescent dyes.In some embodiments, reactions are done in multiplex, e.g., using manyallele-specific primers in the same extension reaction.

In some embodiments, extension products are hybridized to a solid orsemi-solid support, such as beads, matrix, gel, among others. Forexample, the extension products may be tagged with a particular nucleicacid sequence (e.g., included as part of the allele-specific primer) andthe solid support may be attached to an “anti-tag” (e.g., a nucleic acidsequence complementary to the tag in the extension product). Extensionproducts can be captured and detected on the solid support. For example,beads may be sorted and detected. One such system that can be employedin this manner is the LUMINEX™ MAP system, which can be adapted forcystic fibrosis mutation detection by TM Bioscience and is soldcommercially as a universal bead array (TAG-IT™)

Single Nucleotide Primer Extension

In some embodiments, a single nucleotide primer extension (SNuPE) assayis used, in which the primer is designed to be extended by only onenucleotide. In such methods, the identity of the nucleotide justdownstream of the 3′ end of the primer is known and differs in themutant allele as compared to the wild-type allele. SNuPE can beperformed using an extension reaction in which the only one particularkind of deoxynucleotide is labeled (e.g., labeled dATP, labeled dCTP,labeled dGTP, or labeled dTTP). Thus, the presence of a detectableextension product can be used as an indication of the identity of thenucleotide at the position of interest (e.g., the position justdownstream of the 3′ end of the primer), and thus as an indication ofthe presence or absence of a mutation at that position. SNuPE can beperformed as described in U.S. Pat. Nos. 5,888,819; 5,846,710;6,280,947; 6,482,595; 6,503,718; 6,919,174; Piggee, C. et al. Journal ofChromatography A 781 (1997), p. 367-375 (“Capillary Electrophoresis forthe Detection of Known Point Mutations by Single-Nucleotide PrimerExtension and Laser-Induced Fluorescence Detection”); Hoogendoom, B. etal., Human Genetics (1999) 104:89-93, (“Genotyping Single NucleotidePolymorphism by Primer Extension and High Performance LiquidChromatography”), the entire contents of each of which are hereinincorporated by reference.

In some embodiments, primer extension can be combined with massspectrometry for accurate and fast detection of the presence or absenceof a mutation. See, U.S. Pat. No. 5,885,775 to Haff et al. (analysis ofsingle nucleotide polymorphism analysis by mass spectrometry); U.S. Pat.No. 7,501,251 to Koster (DNA diagnosis based on mass spectrometry); theteachings of both of which are incorporated herein by reference.Suitable mass spectrometric format includes, but is not limited to,Matrix-Assisted Laser Desorption/Ionization, Time-of-Flight (MALDI-TOF),Electrospray (ES), IR-MALDI, Ion Cyclotron Resonance (ICR), FourierTransform, and combinations thereof.

Oligonucleotide Ligation Assay

In some embodiments, an oligonucleotide ligation assay (“OLA” or “OL”)is used. OLA employs two oligonucleotides that are designed to becapable of hybridizing to abutting sequences of a single strand of atarget molecules. Typically, one of the oligonucleotides isbiotinylated, and the other is detectably labeled, e.g., with astreptavidin-conjugated fluorescent moiety. If the precise complementarysequence is found in a target molecule, the oligonucleotides willhybridize such that their termini abut, and create a ligation substratethat can be captured and detected. See e.g., Nickerson et al. (1990)Proc. Natl. Acad. Sci. U.S.A. 87:8923-8927, Landegren, U. et al. (1988)Science 241:1077-1080 and U.S. Pat. No. 4,998,617, the entire contentsof which are herein incorporated by reference in their entirety.

Hybridization Approach

In some embodiments, nucleic acids are analyzed by hybridization usingone or more oligonucleotide probes specific for the biomarker ofinterest and under conditions sufficiently stringent to disallow asingle nucleotide mismatch. In certain embodiments, suitable nucleicacid probes can distinguish between a normal gene and a mutant gene.Thus, for example, one of ordinary skill in the art could use probes ofthe invention to determine whether an individual is homozygous orheterozygous for a particular allele.

Nucleic acid hybridization techniques are well known in the art. Thoseskilled in the art understand how to estimate and adjust the stringencyof hybridization conditions such that sequences having at least adesired level of complementary will stably hybridize, while those havinglower complementary will not. For examples of hybridization conditionsand parameters, see, e.g., Sambrook, et al., 1989, Molecular Cloning: ALaboratory Manual, Second Edition, Cold Spring Harbor Press, Plainview,N.Y.; Ausubel, F. M. et al. 1994, Current Protocols in MolecularBiology. John Wiley & Sons, Secaucus, N.J.

In some embodiments, probe molecules that hybridize to the mutant orwildtype sequences can be used for detecting such sequences in theamplified product by solution phase or, more preferably, solid phasehybridization. Solid phase hybridization can be achieved, for example,by attaching probes to a microchip.

Nucleic acid probes may comprise ribonucleic acids and/ordeoxyribonucleic acids. In some embodiments, provided nucleic acidprobes are oligonucleotides (i.e., “oligonucleotide probes”). Generally,oligonucleotide probes are long enough to bind specifically to ahomologous region of the gene of interest, but short enough such that adifference of one nucleotide between the probe and the nucleic acidsample being tested disrupts hybridization. Typically, the sizes ofoligonucleotide probes vary from approximately 10 to 100 nucleotides. Insome embodiments, oligonucleotide probes vary from 15 to 90, 15 to 80,15 to 70, 15 to 60, 15 to 50, 15 to 40, 15 to 35, 15 to 30, 18 to 30, or18 to 26 nucleotides in length. As appreciated by those of ordinaryskill in the art, the optimal length of an oligonucleotide probe maydepend on the particular methods and/or conditions in which theoligonucleotide probe may be employed.

In some embodiments, nucleic acid probes are useful as primers, e.g.,for nucleic acid amplification and/or extension reactions. For example,in certain embodiments, the gene sequence being evaluated for a variantcomprises the exon sequences. In certain embodiments, the exon sequenceand additional flanking sequence (e.g., about 5, 10, 15, 20, 25, 30, 35,40, 45, 50, 55 or more nucleotides of UTR and/or intron sequence) isanalyzed in the assay. Or, intron sequences or other non-coding regionsmay be evaluated for potentially deleterious mutations. Or, portions ofthese sequences may be used. Such variant gene sequences may includesequences having at least one of the mutations as described herein.

Other embodiments of the disclosure provide isolated gene sequencescontaining mutations that relate to the syndrome and/or disease ofinterest. Such gene sequences may be used to objectively diagnose thepresence or increased risk for a subject to develop NCGS. In certainembodiments, the isolated nucleic acid may contain a non-variantsequence or a variant sequence of any one or combination thereof. Forexample, in certain embodiments, the gene sequence comprises the exonsequences. In certain embodiments, the exon sequence and additionalflanking sequence (e.g., about 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55or more nucleotides of UTR and/or intron sequence) is analyzed in theassay. Or, intron sequences or other non-coding regions may be used. Or,portions of these sequences may be used. In certain embodiments, thegene sequence comprises an exon sequence from at least one of thebiomarker genes disclosed herein.

In some embodiments, nucleic acid probes are labeled with a detectablemoiety as described herein.

Arrays

A variety of the methods mentioned herein may be adapted for use witharrays that allow sets of biomarkers to be analyzed and/or detected in asingle experiment. For example, multiple mutations that comprisebiomarkers can be analyzed at the same time. In particular, methods thatinvolve use of nucleic acid reagents (e.g., probes, primers,oligonucleotides, etc.) are particularly amenable for adaptation to anarray-based platform (e.g., microarray). In some embodiments, an arraycontaining one or more probes specific for detecting mutations in thebiomarker of interest.

DNA Sequencing

In certain embodiments, diagnosis of the biomarker of interest iscarried out by detecting variation in the sequence, genomic location orarrangement, and/or genomic copy number of a nucleic acid or a panel ofnucleic acids by nucleic acid sequencing.

In some embodiments, the method may comprise obtaining a nucleic acidfrom a tissue or body fluid sample from a subject and sequencing atleast a portion of a nucleic acid in order to obtain a sample nucleicacid sequence for at least one gene. In certain embodiments, the methodmay comprise comparing the variant to known variants associated withNCGS and determining whether the variant is a variant that has beenpreviously identified as being associated with NCGS. Or, the method maycomprise identifying the variant as a new, previously uncharacterizedvariant. If the variant is a new variant, or in some cases forpreviously characterized (i.e., identified) variants, the method mayfurther comprise performing an analysis to determine whether themutation is expected to be deleterious to expression of the gene and/orthe function of the protein encoded by the gene. The method may furthercomprise using the variant profile (i.e., a compilation of variantsidentified in the subject) to diagnose the presence of NCGS or anincreased risk of developing NCGS.

For example, in certain embodiments, next generation (massively-parallelsequencing) may be used. Or, Sanger sequencing may be used. Or, acombination of next-generation (massively-parallel sequencing) andSanger sequencing may be used. Additionally and/or alternatively, thesequencing comprises at least one of single-moleculesequencing-by-synthesis. Thus, in certain embodiments, a plurality ofDNA samples are analyzed in a pool to identify samples that show avariation. Additionally or alternatively, in certain embodiments, aplurality of DNA samples are analyzed in a plurality of pools toidentify an individual sample that shows the same variation in at leasttwo pools.

One conventional method to perform sequencing is by chain terminationand gel separation, as described by Sanger et al., 1977, Proc Natl AcadSci USA, 74:5463-67. Another conventional sequencing method involveschemical degradation of nucleic acid fragments. See, Maxam et al., 1977,Proc. Natl. Acad. Sci., 74:560-564. Also, methods have been developedbased upon sequencing by hybridization. See, e.g., Harris et al., U.S.Patent Application Publication No. 20090156412. Each of these referencesare incorporated by reference in there entireties herein.

In other embodiments, sequencing of the nucleic acid is accomplished bymassively parallel sequencing (also known as “next generationsequencing”) of single-molecules or groups of largely identicalmolecules derived from single molecules by amplification through amethod such as PCR. Massively parallel sequencing is shown for examplein Lapidus et al., U.S. Pat. No. 7,169,560, Quake et al. U.S. Pat. No.6,818,395, Harris U.S. Pat. No. 7,282,337 and Braslavsky, et al., PNAS(USA), 100: 3960-3964 (2003), the contents of each of which areincorporated by reference herein.

In next generation sequencing, PCR or whole genome amplification can beperformed on the nucleic acid in order to obtain a sufficient amount ofnucleic acid for analysis. In some forms of next generation sequencing,no amplification is required because the method is capable of evaluatingDNA sequences from unamplified DNA. Once determined, the sequence and/orgenomic arrangement and/or genomic copy number of the nucleic acid fromthe test sample is compared to a standard reference derived from one ormore individuals not known to suffer from NCGS at the time their samplewas taken. All differences between the sequence and/or genomicarrangement and/or genomic arrangement and/or copy number of the nucleicacid from the test sample and the standard reference are consideredvariants.

In next generation (massively parallel sequencing), all regions ofinterest are sequenced together, and the origin of each sequence read isdetermined by comparison (alignment) to a reference sequence. Theregions of interest can be enriched together in one reaction, or theycan be enriched separately and then combined before sequencing. Incertain embodiments, and as described in more detail in the examplesherein, the DNA sequences derived from coding exons of genes included inthe assay are enriched by bulk hybridization of randomly fragmentedgenomic DNA to specific RNA probes. The same adapter sequences areattached to the ends of all fragments, allowing enrichment of allhybridization-captured fragments by PCR with one primer pair in onereaction. Regions that are less efficiently captured by hybridizationare amplified by PCR with specific primers. In addition, PCR withspecific primers is may be used to amplify exons for which similarsequences (“pseudo exons”) exist elsewhere in the genome.

In certain embodiments where massively parallel sequencing is used, PCRproducts are concatenated to form long stretches of DNA, which aresheared into short fragments (e.g., by acoustic energy). This stepensures that the fragment ends are distributed throughout the regions ofinterest. Subsequently, a stretch of dA nucleotides is added to the 3′end of each fragment, which allows the fragments to bind to a planarsurface coated with oligo(dT) primers (the “flow cell”). Each fragmentmay then be sequenced by extending the oligo(dT) primer withfluorescently-labeled nucleotides. During each sequencing cycle, onlyone type of nucleotide (A, G, T, or C) is added, and only one nucleotideis allowed to be incorporated through use of chain terminatingnucleotides. For example, during the 1st sequencing cycle, afluorescently labeled dCTP could be added. This nucleotide will only beincorporated into those growing complementary DNA strands that need a Cas the next nucleotide. After each sequencing cycle, an image of theflow cell is taken to determine which fragment was extended. DNA strandsthat have incorporated a C will emit light, while DNA strands that havenot incorporated a C will appear dark. Chain termination is reversed tomake the growing DNA strands extendible again, and the process isrepeated for a total of 120 cycles.

The images are converted into strings of bases, commonly referred to as“reads,” which recapitulate the 3′ terminal 25 to 60 bases of eachfragment. The reads are then compared to the reference sequence for theDNA that was analyzed. Since any given string of 25 bases typically onlyoccurs once in the human genome, most reads can be “aligned” to onespecific place in the human genome. Finally, a consensus sequence ofeach genomic region may be built from the available reads and comparedto the exact sequence of the reference at that position. Any differencesbetween the consensus sequence and the reference are called as sequencevariants.

Detectable Moieties

In certain embodiments, certain molecules (e.g., nucleic acid probes,antibodies, etc.) used in accordance with and/or provided by theinvention comprise one or more detectable entities or moieties, i.e.,such molecules are “labeled” with such entities or moieties.

Any of a wide variety of detectable agents can be used in the practiceof the disclosure. Suitable detectable agents include, but are notlimited to: various ligands, radionuclides; fluorescent dyes;chemiluminescent agents (such as, for example, acridinum esters,stabilized dioxetanes, and the like); bioluminescent agents; spectrallyresolvable inorganic fluorescent semiconductors nanocrystals (i.e.,quantum dots); microparticles; metal nanoparticles (e.g., gold, silver,copper, platinum, etc.); nanoclusters; paramagnetic metal ions; enzymes;colorimetric labels (such as, for example, dyes, colloidal gold, and thelike); biotin; dioxigenin; haptens; and proteins for which antisera ormonoclonal antibodies are available.

In some embodiments, the detectable moiety is biotin. Biotin can bebound to avidins (such as streptavidin), which are typically conjugated(directly or indirectly) to other moieties (e.g., fluorescent moieties)that are detectable themselves.

Below are described some non-limiting examples of some detectablemoieties that may be used.

Fluorescent Dyes

In certain embodiments, a detectable moiety is a fluorescent dye.Numerous known fluorescent dyes of a wide variety of chemical structuresand physical characteristics are suitable for use in the practice of thedisclosure. A fluorescent detectable moiety can be stimulated by a laserwith the emitted light captured by a detector. The detector can be acharge-coupled device (CCD) or a confocal microscope, which records itsintensity.

Suitable fluorescent dyes include, but are not limited to, fluoresceinand fluorescein dyes (e.g., fluorescein isothiocyanine or FITC,naphthofluorescein, 4′,5′-dichloro-2′,7′-dimethoxyfluorescein,6-carboxyfluorescein or FAM, etc.), carbocyanine, merocyanine, styryldyes, oxonol dyes, phycoerythrin, erythrosin, eosin, rhodamine dyes(e.g., carboxytetramethylrhodamine or TAMRA, carboxyrhodamine 6G,carboxy-X-rhodamine (ROX), lissamine rhodamine B, rhodamine 6G,rhodamine Green, rhodamine Red, tetramethylrhodamine (TMR), etc.),coumarin and coumarin dyes (e.g., methoxycoumarin, dialkylaminocoumarin,hydroxycoumarin, aminomethylcoumarin (AMCA), etc.), Q-DOTS. Oregon GreenDyes (e.g., Oregon Green 488, Oregon Green 500, Oregon Green 514, etc.),Texas Red, Texas Red-X, SPECTRUM RED™, SPECTRUM GREEN™, cyanine dyes(e.g., CY-3™, CY-5™, CY-3.5™, CY-5.5™, etc.), ALEXA FLUOR™ dyes (e.g.,ALEXA FLUOR™ 350, ALEXA FLUOR™ 488, ALEXA FLUOR™ 532, ALEXA FLUOR™ 546,ALEXA FLUOR™ 568, ALEXA FLUOR™ 594, ALEXA FLUOR™ 633, ALEXA FLUOR™ 660,ALEXA FLUOR™ 680, etc.), BODIPY™ dyes (e.g., BODIPY™ FL, BODIPY™ R6G,BODIPY™ TMR, BODIPY™ TR, BODIPY™ 530/550, BODIPY™ 558/568, BODIPY™564/570, BODIPY™ 576/589, BODIPY™ 581/591, BODIPY™ 630/650, BODIPY™650/665, etc.), IRDyes (e.g., IRD40, IRD 700, IRD 800, etc.), and thelike. For more examples of suitable fluorescent dyes and methods forcoupling fluorescent dyes to other chemical entities such as proteinsand peptides, see, for example, “The Handbook of Fluorescent Probes andResearch Products”, 9th Ed., Molecular Probes, Inc., Eugene, Oreg.Favorable properties of fluorescent labeling agents include high molarabsorption coefficient, high fluorescence quantum yield, andphotostability. In some embodiments, labeling fluorophores exhibitabsorption and emission wavelengths in the visible (i.e., between 400and 750 nm) rather than in the ultraviolet range of the spectrum (i.e.,lower than 400 nm).

A detectable moiety may include more than one chemical entity such as influorescent resonance energy transfer (FRET). Resonance transfer resultsan overall enhancement of the emission intensity. For instance, see Juet. al. (1995) Proc. Nat'l Acad. Sci. (USA) 92:4347, the entire contentsof which are herein incorporated by reference. To achieve resonanceenergy transfer, the first fluorescent molecule (the “donor” fluor)absorbs light and transfers it through the resonance of excitedelectrons to the second fluorescent molecule (the “acceptor” fluor). Inone approach, both the donor and acceptor dyes can be linked togetherand attached to the oligo primer. Methods to link donor and acceptordyes to a nucleic acid have been described, for example, in U.S. Pat.No. 5,945,526 to Lee et al., the entire contents of which are hereinincorporated by reference. Donor/acceptor pairs of dyes that can be usedinclude, for example, fluorescein/tetramethylrohdamine,IAEDANS/fluroescein, EDANS/DABCYL, fluorescein/fluorescein, BODIPYFL/BODIPY FL, and Fluorescein/QSY 7 dye. See, e.g., U.S. Pat. No.5,945,526 to Lee et al. Many of these dyes also are commerciallyavailable, for instance, from Molecular Probes Inc. (Eugene, Oreg.).Suitable donor fluorophores include 6-carboxyfluorescein (FAM),tetrachloro-6-carboxyfluorescein (TET),2′-chloro-7′-phenyl-1,4-dichloro-6-carboxyfluorescein (VIC), and thelike.

Enzymes

In certain embodiments, a detectable moiety is an enzyme. Examples ofsuitable enzymes include, but are not limited to, those used in anELISA, e.g., horseradish peroxidase, beta-galactosidase, luciferase,alkaline phosphatase, etc. Other examples include beta-glucuronidase,beta-D-glucosidase, urease, glucose oxidase, etc. An enzyme may beconjugated to a molecule using a linker group such as a carbodiimide, adiisocyanate, a glutaraldehyde, and the like.

Radioactive Isotopes

In certain embodiments, a detectable moiety is a radioactive isotope.For example, a molecule may be isotopically-labeled (i.e., may containone or more atoms that have been replaced by an atom having an atomicmass or mass number different from the atomic mass or mass numberusually found in nature) or an isotope may be attached to the molecule.Non-limiting examples of isotopes that can be incorporated intomolecules include isotopes of hydrogen, carbon, fluorine, phosphorous,copper, gallium, yttrium, technetium, indium, iodine, rhenium, thallium,bismuth, astatine, samarium, and lutetium (i.e., 3H, 13C, 14C, 18F, 19F,32P, 35S, 64Cu, 67Cu, 67Ga, 90Y, 99mTc, 111In, 125I, 123I, 129I, 131I,135I, 186Re, 187Re, 201Tl, 212Bi, 213Bi, 211At, 153Sm, 177Lu).

In some embodiments, signal amplification is achieved using labeleddendrimers as the detectable moiety (see, e.g., Physiol Genomics3:93-99, 2000), the entire contents of which are herein incorporated byreference in their entirety. Fluorescently labeled dendrimers areavailable from Genisphere (Montvale, N.J.). These may be chemicallyconjugated to the oligonucleotide primers by methods known in the art.

Kits

In certain embodiments, the disclosure provides kits for use inaccordance with methods and compositions disclosed herein. Generally,kits comprise one or more reagents detect the biomarker of interest.Suitable reagents may include nucleic acid probes and/or antibodies orfragments thereof. In some embodiments, suitable reagents are providedin a form of an array such as a microarray or a mutation panel.

In some embodiments, provided kits further comprise reagents for carriedout various detection methods described herein (e.g., sequencing,hybridization, primer extension, multiplex ASPE, immunoassays, etc.).For example, kits may optionally contain buffers, enzymes, and/orreagents for use in methods described herein, e.g., for amplifyingnucleic acids via primer-directed amplification, for performing ELISAexperiments, etc.

In some embodiments, provided kits further comprise a control indicativeof a healthy individual, e.g., a nucleic acid and/or protein sample froman individual who does not have the disease and/or syndrome of interest.In an embodiment, the control is derived from is a healthy individual orindividuals with no detected or detectable gastrointestinal pathology.In some embodiments, the control is a disease control. Such diseasecontrols may include individuals with coeliac disease (stratified bywhether the individual is on a gluten-free diet or not on a gluten freediet) and subjects with other GI diseases such as inflammatory boweldisease (IBD), hepatitis, small intestinal bacterial overgrowth, andother diseases.

Kits may also contain instructions on how to determine if an individualhas the disease and/or syndrome of interest, or is at risk of developingthe disease and/or syndrome of interest.

In some embodiments, provided is a computer readable medium encodinginformation corresponding to the biomarker of interest. Such computerreadable medium may be included in a kit of the invention.

Methods to Identify NCGS Markers

Data Mining

In certain embodiments of the disclosure, biomarkers are identifiedusing a data mining approach. For example, in some cases publicdatabases (e.g., PubMed) may be searched for genes that have been shownto be linked to (directly or indirectly) to a certain disease. Suchgenes may then be evaluated as biomarkers. In one embodiment, theresults of such an analysis identify IP-10, IL-8, IgE, TNF-alpha, IL-10,CD4 and CD45 as potential markers of interest. FIG. 1 shows an exampleof a multi-node interaction network identifying markers associated withNCGS, as described in detail in U.S. Provisional Patent Application No.62/505,536, filed May 12, 2017 and U.S. Provisional Patent Application62/523,382, filed Jun. 22, 2017, and incorporated by reference in theirentireties herein. In the FIGURE, the circled markers comprise the morerelevant disease markers.

Molecular

In certain embodiments, the disclosure comprises methods to identifybiomarkers for a syndrome or disease of interest (i.e., variants innucleic acid sequence that are associated with NCGS in a statisticallysignificant manner). The genes and/or genomic regions assayed for newmarkers may be selected based upon their importance in biochemicalpathways that show genetic linkage and/or biological causation to thesyndrome and/or disease of interest. Or, the genes and/or genomicregions assayed for markers may be selected based on genetic linkage toDNA regions that are genetically linked to the inheritance of NCGS infamilies. Or, the genes and/or genomic regions assayed for markers maybe evaluated systematically to cover certain regions of chromosomes notyet evaluated.

In other embodiments, the genes or genomic regions evaluated for newmarkers may be part of a biochemical pathway that may be linked to thedevelopment of the syndrome and/or disease of interest (e.g., NCGS). Thevariants and/or variant combinations may be assessed for their clinicalsignificance based on one or more of the following methods. If a variantor a variant combination is reported or known to occur more often innucleic acid from subjects with, than in subjects without, the syndromeand/or disease of interest it is considered to be at least potentiallypredisposing to the syndrome and/or disease of interest. If a variant ora variant combination is reported or known to be transmitted exclusivelyor preferentially to individuals having the syndrome and/or disease ofinterest, it is considered to be at least potentially predisposing tothe syndrome and/or disease of interest. Conversely, if a variant isfound in both populations at a similar frequency, it is less likely tobe associated with the development of the syndrome and/or disease ofinterest.

If a variant or a variant combination is reported or known to have anoverall deleterious effect on the function of a protein or a biologicalsystem in an experimental model system appropriate for measuring thefunction of this protein or this biological system, and if this variantor variant combination affects a gene or genes known to be associatedwith the syndrome and/or disease of interest, it is considered to be atleast potentially predisposing to the syndrome and/or disease ofinterest. For example, if a variant or a variant combination ispredicted to have an overall deleterious effect on a protein or geneexpression (i.e., resulting in a nonsense mutation, a frameshiftmutation, or a splice site mutation, or even a missense mutation), basedon the predicted effect on the sequence and/or the structure of aprotein or a nucleic acid, and if this variant or variant combinationaffects a gene or genes known to be associated with the syndrome and/ordisease of interest, it is considered to be at least potentiallypredisposing to the syndrome and/or disease of interest.

Also, in certain embodiments, the overall number of variants may beimportant. If, in the test sample, a variant or several variants aredetected that are, individually or in combination, assessed as at leastprobably associated with the syndrome and/or disease of interest, thenthe individual in whose genetic material this variant or these variantswere detected can be diagnosed as being affected with or at high risk ofdeveloping the syndrome and/or disease of interest.

For example, the disclosure herein provides methods for diagnosing thepresence or an increased risk of developing NCGS in a subject. Suchmethods may include obtaining a nucleic acid from a sample of tissue orbody fluid. The method may further include sequencing the nucleic acidor determining the genomic arrangement or copy number of the nucleicacid to detect whether there is a variant or variants in the nucleicacid sequence or genomic arrangement or copy number. The method mayfurther include the steps of assessing the clinical significance of avariant or variants. Such analysis may include an evaluation of theextent of association of the variant sequence in affected populations(i.e., subjects having the disease). Such analysis may also include ananalysis of the extent of the effect the mutation may have on geneexpression and/or protein function. The method may also includediagnosis the presence or an increased risk of developing NCGS based onthe assessment.

The following examples serve to illustrate certain aspects of thedisclosure. These examples are in no way intended to be limiting.

Examples Tumor Necrosis Factor Alpha (TNF-α)

Tumor Necrosis Factor alpha (TNF-α), also known as cachectin andTNFSF1A, is the prototypic ligand of the TNF superfamily. TNF-α plays acentral role in inflammation, immune system development, apoptosis, andlipid metabolism. TNF-α is also involved in a number of pathologicalconditions including asthma, Crohn's disease, rheumatoid arthritis,neuropathic pain, obesity, type 2 diabetes, septic shock, autoimmunity,and cancer.

TNF-α may be measured using the Quantikine assay. The Quantikine TNF-αimmunoassay is a 4.0 hour solid phase ELISA designed to measure humanTNF-α in serum and plasma. The assay contains E. coli-derivedrecombinant human TNF-α and antibodies raised against the recombinantfactor, and employs the quantitative sandwich enzyme immunoassaytechnique. A monoclonal antibody specific for human TNF-α is pre-coatedonto a microplate. Standards and samples are pipetted into the wells andTNF-α present is bound by the immobilized antibody. After washing awayany unbound substances, a biotinylated polyclonal antibody specific forhuman TNF-α is added to the wells, the wells are washed to removeunbound antibody-biotin reagent, and an enzyme-linked streptavidin isadded to the wells. After washing, a substrate solution (hydrogenperoxide and tetramethylbenzidine) is added to the wells and colordevelops in proportion to the amount of TNF-α bound in the initial step.The color development is stopped and the intensity of the color ismeasured at 450 nm, subtracting readings at 540 nm and 570 nm.

The samples may be serum or plasma. For serum, a serum separator tube(SST) is used and samples are allowed to clot for 30 minutes at roomtemperature before centrifugation for 15 minutes at 1000×g. The serum isremoved and assayed. Samples are used immediately or aliquoted andstored at ≤−20° C. Plasma is collected using EDTA or heparin as ananticoagulant. The samples are centrifuged for 15 minutes at 1000×gwithin 30 minutes of collection, and samples assayed immediately oraliquoted and stored at ≤−20° C. For both serum and plasma samples,repeated freeze-thaw cycles should be avoided. Generally, grosslyhemolyzed samples, high albumin samples, and citrate plasma should notbe used.

Interleukin 10 (IL-10)

Interleukin 10 (IL-10), initially designated cytokine synthesisinhibitory factor (CSIF). IL-10 is a pleiotropic cytokine that can exerteither immunosuppressive or immunostimulatory effects on a variety ofcell types. IL-10 is a potent modulator of monocyte/macrophage function.As a down-regulator of the cell-mediated immune response, IL-10 cansuppress the production of prostaglandin E2 and numerous proinflammatorycytokines, including TNF-alpha, IL-1, IL-6, and LL-8 by monocytesfollowing activation. IL-10 also enhances the release of soluble TNFreceptors and inhibits the expression of surface ICAM-1 and B. IL-10 hasbeen reported to suppress the synthesis of superoxide anion plusreactive oxygen intermediates (ROI), and either inhibit or facilitate NOsynthesis, depending on the time of exposure to activated macrophages.IL-10 also has marked effects on B cells. For example, it induces IgAsynthesis in CD40-activated cells and selects for the secretion of IgG1and IgG3. IL-10 also has documented activity on endothelial cells, whereit mimics IL-4, and on thymocytes and mast cells, where it acts as agrowth co-stimulator.

IL-10 may be measured using the Quantikine assay. The Quantikine IL-10Immunoassay is a 3.5-4.5 hour solid phase ELISA designed to measureIL-10 in cell culture supernatants, serum, and plasma. It contains Sf21-expressed recombinant human IL-10 and antibodies raised against therecombinant factor. The assay employs a quantitative sandwich enzymeimmunoassay technique. A monoclonal antibody specific for IL-10 ispre-coated onto a microplate. Standards and samples are pipetted intothe wells and any LL-10 present is bound by the immobilized antibody.After washing away any unbound substances, an enzyme-linked monoclonalantibody specific for IL-10 is added to the wells. After washing, asubstrate solution (hydrogen peroxide and tetramethylbenzidine) is addedto the wells and color develops in proportion to the amount of TNF-αbound in the initial step. The color development is stopped and theintensity of the color is measured at 450 nm, subtracting readings at540 nm and 570 nm.

For cell culture supernatants, particulates are removed bycentrifugation. Samples are assayed immediately or aliquoted and storedat ≤−20° C. Repeated freeze-thaw cycles should be avoided. For serum, aserum separator tube (SST) is used and samples are allowed to clot for30 minutes at room temperature before centrifugation for 15 minutes at1000×g. The serum is removed and assayed. Samples are used immediatelyor aliquoted and stored at ≤−20° C. Plasma is collected using EDTA orheparin as an anticoagulant. The samples are centrifuged for 15 minutesat 1000×g within 30 minutes of collection, and samples assayedimmediately or aliquoted and stored samples at ≤−20° C. For both serumand plasma samples, repeated freeze-thaw cycles should be avoided.Generally, grossly hemolyzed samples and high albumin samples should notbe used.

Interleukin 8 (IL-8)

Interleukin 8 (IL-8), a member of the neutrophil-specific CXC subfamilyof chemokines, is a potent neutrophil chemotactic and activating factor.It is a primary inflammatory cytokine produced by many cells (includingmonocytes/macrophages, T cells, neutrophils, fibroblasts, endothelialcells, keratinocytes, hepatocytes, astrocytes and chondrocytes) inresponse to proinflammatory stimuli such as IL-1, TNF, LPS and viruses.Its function is, in part, to attract neutrophils to the site ofinflammation and to activate them. IL-8 binds to twoseven-transmembrane, G protein-coupled receptors, CXCR1 and CXCR2, aswell as to the non-signalling Duffy antigen on red blood cells. TheDuffy antigen may play a role in regulating IL-8 activity on functionalreceptors.

IL-8 can be measured using the Quantikine assay. The Quantikine IL-8immunoassay is a 3.5 hour solid phase ELISA designed to measure humanIL-8 in cell culture supernatants, serum, and plasma. It is based onantibodies raised against the 72 amino acid variant of human IL-8derived from E. coli. It is calibrated with the same recombinant factor.The assay employs the quantitative sandwich enzyme immunoassaytechnique. A monoclonal antibody specific for IL-8 has been pre-coatedonto a microplate. Standards and samples are pipetted into the wells andany IL-8 present is bound by the immobilized antibody. After washingaway any unbound substances, an enzyme-linked monoclonal antibodyspecific for IL-8 is added to the wells. After washing, a substratesolution (hydrogen peroxide and tetramethylbenzidine) is added to thewells and color develops in proportion to the amount of TNF-α bound inthe initial step. The color development is stopped and the intensity ofthe color is measured at 450 nm, subtracting readings at 540 nm and 570nm.

For cell culture supernatants, particulates are removed bycentrifugation. Samples are assayed immediately or aliquoted and storedat ≤−20° C. Repeated freeze-thaw cycles should be avoided. For serum, aserum separator tube (SST) is used and samples are allowed to clot for30 minutes at room temperature before centrifugation for 15 minutes at1000×g. The serum is removed and assayed. Samples are used immediatelyor aliquoted and stored at ≤−20° C. Plasma is collected using EDTA orheparin as an anticoagulant. The samples are centrifuged for 15 minutesat 1000×g within 30 minutes of collection, and samples assayedimmediately or aliquoted and stored at ≤−20° C. For both serum andplasma samples, repeated freeze-thaw cycles should be avoided.Generally, grossly hemolyzed samples and high albumin samples should notbe used.

Total IgE

Immunoglobulin E (IgE) plays an important role in immunologicalprotection against parasitic infections and in allergy (type 1hypersensitivity). For example, the binding of the allergen tosensitized mast cells or basophilic cells can result in cross-linking ofIgE on the cell membrane, leading to cell degranulation and the releaseof factors (e.g. histamine), which produce the typical symptoms of type1 hypersensitivity. The IgE concentration in serum is normally very low(≤0.001% of the total serum immunoglobulin). The IgE concentration isgenerally age-dependent, with the lowest values being measured at birth.Its concentration gradually increases and becomes stabilized between theage of 5-7, although the IgE values can vary greatly within particularage groups. In infants and small children with recurrent respiratorytract diseases, the determination of IgE can be of prognostic relevance.As IgE is of importance in allergies, elevated IgE concentrations can befound in patients with allergic diseases such as hay fever, atopicbronchitis and dermatitis. Elevated serum IgE concentrations can alsooccur in non-allergic diseases, e.g. bronchopulmonary aspergillosis,Wiskott-Aldrich syndrome, hyper-IgE syndrome, IgE myeloma, and parasiticinfections.

The IgE II assay (Elecsys) uses monoclonal antibodies specificallydirected against human IgE. The Elecsys assay is a sandwich immunoassay.During the first incubation, IgE in the sample (10 μL) is mixed with abiotinylated monoclonal IgE-specific antibody, and a monoclonalIgE-specific antibody labeled with a ruthenium complex(Tris(2,2′-bipyridyl)ruthenium(II)-complex (Ru(bpy)2+) to form asandwich complex. After addition of streptavidin-coated microparticles,the complex becomes bound to the solid phase via interaction of biotinand streptavidin. The reaction mixture is then aspirated into themeasuring cell where the microparticles are magnetically captured ontothe surface of the electrode. Unbound substances are then removed withProCell. Application of a voltage to the electrode induceschemiluminescent emission which is measured by a photomultiplier.Results are determined via a calibration curve which isinstrument-specifically generated by 2-point calibration and a mastercurve provided via the reagent barcode.

Alternatively, an ImmunoCAP based assay (Phadia US, Inc.) may be used.In this assay, anti-IgE, covalently coupled to ImmunoCAP, reacts withthe total IgE in the patient sample. After washing, enzyme labeledantibodies against IgE are added to form a complex. Followingincubation, unbound enzyme-anti-IgE is washed away and the bound complexis then incubated with a developing agent. After stopping the reaction,the fluorescence of the eluate is measured. The fluorescence is directlyproportional to the concentration of IgE in the sample. The higher theresponse, the more IgE is present in the sample. To evaluate the testresults, the responses for the patient samples are transformed toconcentrations with the use of a calibration curve.

A variety of patient samples may be used. Serum may be collected usingstandard sampling tubes or tubes containing separating gel, Li⁺-heparin,Na⁺-heparin, K⁺-EDTA, and sodium citrate plasma. When sodium citrate isused, the results must be corrected by +10%.

References and citations to other documents, such as patents, patentapplications, patent publications, journals, books, papers, webcontents, have been made throughout this disclosure. All such documentsare hereby incorporated herein by reference in their entirety for allpurposes. Various modifications and equivalents of those describedherein, will become apparent to those skilled in the art from the fullcontents of this document, including references to the scientific andpatent literature cited herein. The subject matter herein containsinformation, exemplification and guidance that can be adapted to thepractice of this disclosure in its various embodiments and equivalentsthereof.

That which is claimed is:
 1. A method of measuring biomarkers associated with non-coeliac gluten sensitivity (NCGS) in an individual suspected of having NCGS, comprising: obtaining a sample from the individual suspected of having NCGS; and measuring in the sample obtained from the individual suspected of having NCGS an established statistical difference from a predetermined NCGS control value of an amount of at least one of: interleukin 8 (IL-8), an amount of total immunoglobulin E (IgE), an amount of IL-8 and IL-10, an amount of IL-8 and TNF-Alpha, an amount of IL-8 and total IgE, an amount of IL-10 and total IgE, or an amount of TNF-Alpha and total IgE, wherein the predetermined NCGS control value is an amount of at least one of IL-8, IL-10, TNF-Alpha, and total IgE found in individuals with no NCGS.
 2. The method of claim 1, wherein the measuring the established statistical difference comprises performing at least one immunoassay.
 3. The method of claim 1, further comprising measuring expression of at least one of interferon gamma-induced protein 10 (IP-10), CD4 or CD45 in the sample obtained from the individual suspected of having NCGS relative to control expression value in the individuals with no NCGS.
 4. The method of claim 3, wherein the measuring the expression comprises performing flow cytometry.
 5. The method of claim 1, wherein the sample comprises blood, serum, plasma or a tissue biopsy.
 6. A method of measuring biomarkers associated with non-coeliac gluten sensitivity (NCGS) in an individual suspected of having NCGS, comprising: obtaining a sample from the individual suspected of having NCGS; and, measuring an established statistical difference from a predetermined NCGS control value of at least one of: expression of interleukin 8 (IL-8), expression of total immunoglobulin E (IgE), expression of IL-8 and IL-10, expression of IL-8 and TNF-Alpha, expression of IL-8 and total IgE, expression of IL-10 and total IgE, or expression of TNF-Alpha and total IgE, wherein the predetermined NCGS control value is an expression of least one of IL-8, IL-10, TNF-alpha and total IgE found in individuals with no NCGS.
 7. The method of claim 6, wherein the measuring the established statistical difference comprises performing at least one immunoassay.
 8. The method of claim 6, wherein the measuring the established statistical difference comprises performing flow cytometry.
 9. The method of claim 6, further comprising measuring expression of at least one of interferon gamma-induced protein 10 (IP-10), CD4 or CD45 in the sample obtained from the individual suspected of having NCGS relative to control expression value in the individuals with no NCGS.
 10. The method of claim 9, wherein the measuring the expression comprises performing flow cytometry.
 11. The method of claim 6, wherein the sample comprises blood, serum, plasma or a tissue biopsy.
 12. A composition for measuring biomarkers associated with non-coeliac gluten sensitivity (NCGS) in an individual suspected of having NCGS, comprising reagents for measuring in the sample obtained from the individual suspected of having NCGS an established statistical difference from a predetermined NCGS control value of an amount or expression of at least one of: interleukin 8 (IL-8), an amount of total immunoglobulin E (IgE), an amount of IL-8 and IL-10, an amount of IL-8 and TNF-Alpha, an amount of IL-8 and total IgE, an amount of IL-10 and total IgE, or an amount of TNF-Alpha and total IgE, wherein the predetermined NCGS control value is an amount or expression of at least one of IL-8, IL-10, TNF-Alpha, and total IgE found in individuals with no NCGS.
 13. A kit comprising the composition of claim
 12. 